The present invention relates to pharmaceutical compositions for the treatment of hepatitis B virus (HBV) infection.
HBV infection in humans can cause chronic liver disease which will, in some cases, proceed to hepatocellular carcinoma. The initial steps of HBV attachment to cells and the targeting of the viral genome to the host cell nucleus have yet to be deciphered. The specific receptor for HBV has not so far been identified, even though various serum proteins and cellular membrane glycoproteins have been suggested as mediators of cell penetration or viral receptors. HBV envelope proteins were reported to contain residues which interact with polymerized albumin [P. Pontisso, et al., Journal of Virology, Vol. 63, No. 1981-1, p. 988 (1981)] or with soluble transferring [M. Gagliardi, et al., Eur. J. Immunol., Vol. 24, pp. 1372-1376 (1994)], enabling viral penetration of cells via their respective receptors, probably in a non-specific manner.
In a study reported by Neurath, et al. [A. Neurath, et al., J. Exp. Med., Vol. 175, pp. 461-469 (1992)] hIL-6 was shown to bind the pS1 (aa 21-47) segment of the HBV envelope. Putative candidates for the HBV receptor were recently reported, including Annexin V (endohexin II) [K. Hertogs, et al., Virology, Vol. 197, pp. 549-557 (1993)]; apolipoprotein H [H. Mehdi, et al., Journal of Virology, Vol. 68, pp. 2415-2424 (1994)]; and asialoglycoprotein receptor [U. Treichel, et al., Journal of General Virology, Vol. 75, pp. 3021-3029 (1994)].
Binding experiments have demonstrated that the pre-SI (pS1)region of the viral envelope protein contains a recognition site for the host cell [A.R. Neurath, et al., Cell, Vol. 46, pp. 429-436 (1986); M. Petit, et al., Virology, Vol. 180, pp. 483-491 (1990); M. Petit, et al., Virology, Vol. 197, pp. 211-222 (1992)]. Although previous studies had suggested that HepG2 cells [R. Bchini, et al., Journal of Virology, Vol. 64, pp. 3025-3032 (1991)] and human hepatocytes [P. Gripon, et al., Journal of Virology, Vol. 62, pp. 4136-4143 (1988); T. Ochiya, et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 86, pp. 1875-1879 (1989); P. Gripon, et al., Virology, Vol. 192, pp. 534-540 (1993); P. Galle, et al., Gastroenterology, Vol. 106, pp. 664-673 (1994)] could support HBV infection in vitro, no cellular receptor has as yet been defined in either system, and these models were of low experimental reproducibility.
In current reports, it has been shown that a chimeric mouse, generated by using Beige/Nude/X linked immunodeficient (BNX) mice, preconditioned by total body irradiation (12Gy) and reconstituted with severe combined immunodeficient (SCID) mice bone marrow (BM) cells, is permissive for normal human T and B cells [I. Lubin, et al., Science, Vol. 252, pp. 427-431 (1991)], as well as for normal human liver tissue [E. Galun, et al., Journal of Infectious Diseases, Vol. 175, pp. 25-30 (1995)]. Hepatitis C virus (HCV) viremia was detectable for up to two months, after implantation under the kidney capsule of the BNX greater than SCID chimeric animals of either a human liver fragment with preexisting HCV infection, or normal human liver tissue following incubation ex-vivo of the transplanted liver fragment with HCV-positive sera [E. Galun, et al., ibid.].
Earlier studies have revealed that human interleukin 6 (hIL6) contains recognition sites for the hepatitis B virus (HBV). Chinese hamster ovary cells transfected with human IL-6 cDNA and Spondoptera frugiperdaovarian insect cells infected with recombinant baculovirus carrying human IL-6 cDNA expressed receptors for the preS921-47) region of the HBV envelope protein, indicating that expression of IL-6 sequences encompasses a binding site for the HBV envelope protein. Thus, the possibility of developing antiviral compounds mimicking the receptor binding site for HBV on IL-6 but not displaying undesirable biological effects of the intact IL-6 molecule was raised, because it was found that the interaction between the preS1 region of the HBV envelope proteins and cells of hepatic origin was inhibited by IL-6 and by anti-IL-6 antibodies [A. Neurath, et al., J. Exp. Med. Vol. 176, pp. 1561-1569 (1992)].
Heretofore, one of the major obstacles in elucidating the initial steps of HBV infection and the assessment of antiviral agents, has been the lack of a small animal model. Using the techniques referred to above, it was possible to develop SCID greater than BNX animals which sustain HBV viremia following the implantation of an ex-vivo HBV DNA-positive sera incubation with liver tissue. The method in which the animals were prepared for the experiments described herein, and the surgical technique for transplantation, are similar to those previously reported [E. Galun, et al., ibid.].
As described, e.g., in PCT/US94/05410, it has now been found, using a chimeric animal model, that human interleukin 6 (hIL6) is essential for HBV infection. Having identified that hIL6 serves as an essential bridge for HBV infection, the invention now provides a pharmaceutical composition for the treatment of hepatitis B virus infection, comprising an amount of a soluble active agent which interacts with at least one of the binding sites between hIL6 and pS1 and between hIL6 and hepatocytes and other HBV-permissive cells, said active agent being present in sufficient amount to competitively bind to at least one of said sites and thereby to prevent hIL6-mediated HBV infection of hepatocytes and other HBV-permissive cells.
In a first preferred embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of hepatitis B virus (HBV) infection, comprising an amount of soluble gp80 and/or gp130 receptor sites sufficient to inhibit the binding of hIL6 to hepatocytes and other HBV-permissive cells.
In a second preferred embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of HBV infection, comprising an amount of soluble amino acid sequences corresponding to amino acids 21 to 46 of pS1 to block the interaction of HBV with hIL6.
In a third preferred embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of HBV infection, comprising an amount of a soluble ligand selected from the group consisting of peptides LYS41-ALA56, GLY77-GLU95 and GLN153-HIS165 to block the interaction of hIL6 with hepatocytes and other HBV-permissive cells.
In a fourth preferred embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of HBV infection, comprising hIL6 conjugated with an anti-viral agent. In a preferred embodiment, the anti-viral agent conjugated to hIL6 comprises glycoprotein 80 or a portion thereof. In a preferred embodiment of the invention, the hIL6-anti-viral agent conjugate is Hyper-IL6 (HIL6).
In yet another preferred embodiment of the invention, there is provided a pharmaceutical composition for the treatment of hepatitis B virus (HBV) infection of hepatocytes, comprising a soluble active agent which competitively interacts with at least one of the binding sites between human interleukin 6 (hIL6) and hepatocytes, said soluble active agent being selected from the group consisting of glycoprotein 80 (gp80) having receptor sites which interact with hIL6, soluble glycoprotein 130 (gp130) having receptor sites which interact with hIL6, hIL6 derived peptide LYS41-ALA56, hIL6 derived peptide GLY77-GLU95, hIL6 derived peptide GLN153-HIS165, a combined xcex21 and xcex22 hIL6 mutant (mhIL6xcex21+xcex22), and mhIL6xcex21+xcex22 substituted with phe 171 to leu and ser 177 to arg, and mixtures of any of the foregoing. In certain preferred embodiments, such a pharmaceutical composition includes an effective amount of the soluble active agent to treat infection of the hepatocytes by HBV.
In yet another preferred embodiment of the invention, there is provided a pharmaceutical composition for the treatment of hepatitis B virus (HBV) infection of hepatocytes, comprising a soluble active agent which disrupts the hIL6/hIl6Rxcex1 complex with hIL6Rxcex2.
In yet another preferred embodiment of the invention, there is provided a pharmaceutical composition for the treatment of hepatitis B virus (HBV) infection of hepatocytes, comprising a soluble active agent selected from the group consisting of glycoprotein 80 (gp80) having receptor sites which interact with hIL6, soluble glycoprotein 130 (gpl30) having receptor sites which interact with hIL6, hIL6 derived peptide LYS41-ALA56, hIL6 derived peptide GLY77-GLU95, hIL6 derived peptide GLN153-HIS165, a combined xcex21 and xcex22 hIL6 mutant (mhIL6xcex21+xcex22), mhIL6xcex21+xcex22 substituted with phe 171 to leu and ser 177 to arg, a soluble active agent which disrupts the hIL6/hIl6Rxcex1 complex with hIL6Rxcex2, and mixtures of any of the foregoing.
In general with respect to the pharmaceutical compositions described above, the soluble active agent is included in an amount effective to treat HBV infection of hepatocytes, e.g., the soluble active agent is present in an amount from about 100 ng/kg to about 100 mg/kg, based on the body weight of the patient. In preferred embodiments, such a pharmaceutical composition includes a soluble active agent in an amount from about 10 xcexcg/kg to about 10 mg/kg, based on the body weight of the patient.
The invention is further directed to a method for treatment of infection of hepatocytes with HBV, comprising administering to a human patient a soluble active agent which inhibits the interaction between human interleukin 6 (hIL6) and hepatocytes. In certain preferred embodiments of the method, the active agent competitively interacts with at least one of the binding sites. In such preferred embodiments, it is further preferred that the soluble active agent comprises a soluble glycoprotein 80 (gp80) and/or soluble glycoprotein 130 (gp 130) having receptor sites which bind to hIL6 and competitively inhibit the interaction between hIL6 and hepatocytes. In other preferred embodiments of the method, it is further preferred that the soluble active agent comprises a soluble ligand selected from the group consisting of peptides LYS41-ALA56, GLY77-GLU95 and GLN153-HIS165 and competitively blocks the interaction of hIL6 with hepatocytes. In yet other preferred embodiments of the method, it is further preferred that the soluble active agent disrupts the hIL6/hIl6Rxcex1 complex with hIL6Rxcex2. In other preferred embodiments of the method, it is further preferred that the soluble active agent is selected from the group consisting of hIL6 derived peptide LYS41-ALA56, hIL6 derived peptide GLY77-GLU95, hIL6 k) derived peptide GLN153-HIS165, a combined xcex21 and xcex22 hIL6 mutant (mhIL6xcex21+xcex22), and mhIL6xcex21+xcex22 substituted with phe 171 to leu and ser 177 to arg, and mixtures of any of the foregoing. In any of the preferred methods, it is preferred that the soluble active agent is administered in an amount from about 100 ng/kg to about 100 mg/kg per day, and in certain embodiments from about 10 xcexcg/kg to about 10 mg/kg, based on the body weight of the patient.
The invention is further directed to the use of a soluble active agent which inhibits the interaction between human interleukin 6 (hIL6) and hepatocytes for the treatment of infection of hepatocytes with HBV. In certain preferred embodiments, the active agent competitively interacts with at least one of the binding sites. In such preferred embodiments, it is further preferred that the soluble active agent comprises a soluble glycoprotein 80 (gp80) and/or soluble glycoprotein 130 (gp130) having receptor sites which bind to hIL6 and competitively inhibit the interaction between hIL6 and hepatocytes. Alternatively or in addition thereto, the soluble active agent comprises a soluble ligand selected from the group consisting of peptides LYS41-ALA56, GLY77-GLU95 and GLN153-HIS165 and competitively blocks the interaction of hIL6 with hepatocytes. In yet other preferred embodiments, it is further preferred that the soluble active agent disrupts the hIL6/hIl6Rxcex1 complex with hIL6xcex2. Alternatively or in addition thereto, in other preferred embodiments, it is further preferred that the soluble active agent is selected from the group consisting of hIL6 derived peptide LYS41-ALA56, hIL6 derived peptide GLY77-GLU95, hIL6 derived peptide GLN153-HIS165, a combined xcex21 and xcex22 hIL6 mutant (mhIL6xcex21+xcex22), and mhIL6xcex21+xcex22 substituted with phe 171 to leu and ser 177 to arg, and mixtures of any of the foregoing. Alternatively or in addition to the competitively active soluble active agents identified in this above paragraph, the soluble active agent may comprise anti-viral agents conjugated with hIL-6, which inhibit HBV (e.g. 3TC, Famciclovir and FIAU analogues); a toxin which exerts cellular toxicity only to cells expressing IL-6R (e.g., the chimeric fusion toxin DAB389-IL-6); monoclonal antibodies specific for hIL-6 which bind either at site I or site II on the IL-6 protein; a minibody polypeptide (e.g., MB02) which binds tightly and specifically to hIL-6 and is an effective inhibitor of the cytokine""s biological activity; an anti-IL-6R mAb, and mixtures of any of the foregoing. In any of the embodiments described in this paragraph, it is preferred that the soluble active agent is administered an effective amount to treat HBV infection of the hepatocytes. This effective amount will generally be, e.g., an amount from about 100 ng/kg to about 100 mg/kg per day, and in certain embodiments from about 10 xcexcg/kg to about 10 mg/kg, based on the body weight of the patient. Further, the examples provided in this paragraph are not meant to be exclusive. Many other soluble active agents will be readily apparent to one skilled in the art having the benefit of reading this specification.
For purposes of the present invention, the term hIL-6 is human interleukin 6; the term pS1 stands for pre-S1; gp80 is glycoprotein 80 (also referred to as hIL-6R or hIL-6Rxcex1); and gp130 is glycoprotein 130, otherwise known as hIL-6Rxcex2 (human interleukin 6 receptor beta).
For purposes of the present invention, the terms xe2x80x9cbindxe2x80x9d and xe2x80x9cinteractxe2x80x9d shall be interchangeable.